Tear levels of IL-7, IL-1α, and IL-1ß may differentiate between IgG4-related disease and Sjögren's syndrome.

We aim to assess and compare a cytokine and chemokine profile in tears from patients with IgG4-related disease (IgG4-RD) and Sjögren's syndrome (SS), and to see if this profile could aid in differentiating these two diseases. We included 10 patients with IgG4-RD who met the Comprehensive Diagnostic Criteria for IgG4-RD and 17 patients who met the AECG criteria for primary SS. The Schirmer-I test was carried out using two standardized sterile tear strips, which were then immediately frozen at - 86 °C until assayed. The tears were extracted from the strips after they had been defrosted using a buffer containing 0.5 M NaCl and 0.5% Tween-20. The amounts (pg/ml) of the following cytokines and chemokines were then measured using luminometry: IFN-γ, TNF-α, G-CSF, IL-1-α, IL-1ß, IL-4, IL-7, IL-12p40, IL-12p70, IL-13, IL-17A, CCL2, CCL3, CCL4, CCL11, and CXCL10. In the IgG4-RD group, seven patients had lacrimal gland involvement, five had dry eye symptoms, and six had a positive Schirmer-I test. In the SS group, 16 (94.1%) had dry eyes and all had a positive Schirmer-I test. We were able to differentiate between both diseases using levels of IL-7, IL-1α, and IL-1ß; in particular, the IL-7/IL-1α and IL-7/IL-1ß ratios had the best discriminatory potential, with cut-off values of 0.32 (AUC: 0.93, sensitivity: 94%, specificity: 80%, p = 0.0003) and 12.55 (AUC: 0.96, sensitivity: 94%, specificity: 90%, p = 0.0001), respectively. Our results suggest that IL-7, IL-1α, and IL-1ß tear levels could help differentiate IgG4-RD from SS. Key Points • The lacrimal gland is frequently involved in IgG4-RD and SS. This characteristic makes both diseases mimics of one another. • Patients with IgG4-RD and SS have different profiles of tear cytokines and chemokines. • Tear IL-7, IL-1α, and IL-1ß levels may serve as helpful biomarkers in separating IgG4-RD from SS.

Identification of new IL-7Rα small-molecule agonists: a multi-computational approach.

Interleukin 7 (IL-7) is an essential cytokine that acts as a potent growth factor of T-cells and supports the growth of B-cell precursors. IL-7 binds to a heterodimeric receptor consisting of an IL-7 receptor alpha (IL-7Rα) and the common gamma chain receptor (γc) which is shared with IL-2, IL-4, IL-9, IL-15 and IL-21. The discovery of small-molecule agonists of cytokines would be of great pharmaceutical interest with the increasing scientific rationale. In this study, a series of molecular modelling methods, including field-based pharmacophore virtual screening, protein-protein docking and molecular dynamics simulations, led to the identification of two compounds (i.e. 1 and 2) of different classes that exhibit enhanced agonistic effects by activating the IL-7 signalling cascade. One of these compounds was selected as a hit and represents the first small-molecule agonist of IL-7Rα with single-digit micromolar activity. Moreover, the prediction model of the active compound to the IL-7Rα/γc interaction complex provides insight into the binding of a small-molecule agonist to its receptor.

Modulation of Signaling Mediated by TSLP and IL-7 in Inflammation, Autoimmune Diseases, and Cancer.

Thymic Stromal Lymphopoietin (TSLP) and Interleukin-7 (IL-7) are widely studied cytokines within distinct branches of immunology. On one hand, TSLP is crucially important for mediating type 2 immunity at barrier surfaces and has been linked to widespread allergic and inflammatory diseases of the airways, skin, and gut. On the other hand, IL-7 operates at the foundations of T-cell and innate lymphoid cell (ILC) development and homeostasis and has been associated with cancer. Yet, TSLP and IL-7 are united by key commonalities in their structure and the structural basis of the receptor assemblies they mediate to initiate cellular signaling, in particular their cross-utilization of IL-7Rα. As therapeutic targeting of TSLP and IL-7 via diverse approaches is reaching advanced stages and in light of the plethora of mechanistic and structural data on receptor signaling mediated by the two cytokines, the time is ripe to provide integrated views of such knowledge. Here, we first discuss the major pathophysiological roles of TSLP and IL-7 in autoimmune diseases, inflammation and cancer. Subsequently, we curate structural and mechanistic knowledge about receptor assemblies mediated by the two cytokines. Finally, we review therapeutic avenues targeting TSLP and IL-7 signaling. We envision that such integrated view of the mechanism, structure, and modulation of signaling assemblies mediated by TSLP and IL-7 will enhance and fine-tune the development of more effective and selective approaches to further interrogate the role of TSLP and IL-7 in physiology and disease.

Mechanical Stretching of Fibronectin Fibers Upregulates Binding of Interleukin-7.

Since evidence is rising that extracellular matrix (ECM) fibers might serve as reservoirs for growth factors and cytokines, we investigated the interaction between fibronectin (FN) and interleukin-7 (IL-7), a cytokine of immunological significance and a target of several immunotherapies. By employing a FN fiber stretch assay and Förster resonance energy transfer (FRET) confocal microscopy, we found that stretching of FN fibers increased IL-7 binding. We localized the FN binding site on the CD loop of IL-7, since a synthetic CD loop peptide also bound stronger to stretched than to relaxed FN fibers. On the basis of a structural model, we propose that the CD loop can bind to FN, while IL-7 is bound to its cognate cell surface receptors. Sequence alignment with bacterial adhesins, which also bind the FN N-terminus, suggests that a conserved motif on the CD loop (110TKSLEEN116 and the truncated 112SLEE115 in human and mouse IL-7, respectively) might bind to the second FN type I module (FnI2) and that additional epitopes enhance the stretch-upregulated binding. FN fiber stretching might thus serve as a mechano-regulated mechanism to locally concentrate IL-7 in an ECM-bound state, thereby upregulating the potency of IL-7 signaling. A feedback model mechanism is proposed that could explain the well-known, but poorly understood, function of IL-7 in ECM homeostasis. Understanding how local IL-7 availability and signaling might be modulated by the tensional state of the ECM niche, which is adjusted by residing stroma cells, is highly relevant for basic science but also for advancing IL-7 based immunotherapies.

Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS.

Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.

High salivary soluble L-selectin and interleukin-7 levels in Asian Indian patients with primary Sjögren's syndrome.

In present study, we aimed to study salivary soluble L-selectin (sL-selectin), interleukin-7(IjL-7), and lymphotoxin-α levels in primary Sjögren's syndrome (pSS) and their clinical as well as serological correlations. pSS patients fulfilling either the American European Consensus Group (AECG) and/or the American college of Rheumatology (ACR) criteria were recruited. Age- and sex-matched hospital staff were recruited as healthy controls. Unstimulated saliva was collected by the spitting method; sL-selectin, IL-7, and lymphotoxin-α were measured in the saliva using commercial ELISA kits. Forty-three patients with pSS and 31 healthy controls were included in the study. Increased levels of sL-selectin and IL-7 were found in the saliva of patients as compared to controls. Lymphotoxin-α was undetectable in the saliva of pSS patients and controls. Salivary sL-selectin positively correlated with rheumatoid factor (r = 0.47; p < 0.003). No other variable including ESSDAI was significantly associated with salivary sL-selectin and IL-7 levels. Indian patients with primary Sjögren's syndrome have higher salivary sL-selectin and IL-7 levels than healthy controls.

Molecular cloning of chicken IL-7 and characterization of its antiviral activity against IBDV in vivo.

Mammalian interleukin-7 (IL-7) is able to stimulate lymphocyte proliferation and maturation, and reverse immunosuppression. However, whether poultry IL-7 has similar functions remains unclear. Chicken infectious bursal disease virus (IBDV) causes serious immunosuppression in chicken due to virus-induced immune disorder. Whether chicken IL-7 (chIL-7) has the ability to restore the immunity during IBDV-induced immunosuppression is not clear. To test this, we amplified chIL-7 gene by RT-PCR, prepared recombinant chIL-7 using HEK293T cells and treated the chicken with the chIL-7 prior to IBDV infection. Our results indicate that chIL-7 promoted mouse B cell proliferation in vitro, and significantly reduced virus titer in bursal tissue and chicken morbidity of IBDV-infected chicken. Mechanically, chIL-7 induced chicken lymphocyte proliferation and interferon-γ production, but down-regulated TGF-ß expression, suggesting that chIL-7 has the ability to reverse IBDV-induced immunosuppression and might be a potential therapeutic agent for prevention and treatment of infectious bursal disease.

A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture.

Human interleukin-7 (hIL-7) is a therapeutically important cytokine involved in lymphocyte development and survival. In previous reports, a uniformly poor expression of hIL-7 has been shown in Escherichia coli host with the problem of inclusion body formation. In this study, the role of codon optimization and N-terminus blocking using various solubility enhancer fusion tags was explored to improve its soluble expression. The use of codon optimization strategy improved its expression to 80 ± 5 mg/L at shake flask level. The utilization of pelB leader sequence resulted in an unprocessed protein in the form of cytoplasmic inclusion bodies with lower expression yields. The N-terminus fusion of small ubiquitin-like modifier (SUMO), thioredoxin (Trx), and NusA tags increased the expression in the range of 90-140 mg/L, where >90 % of the fusion protein was obtained in soluble form. The fed-batch fermentation of SUMO-tagged hIL-7 protein was optimized at bioreactor level, where a high volumetric product concentration of 2.65 g/L was achieved by controlling the plasmid segregation instability using high antibiotic concentration. The specific product yield (YP/X) and volumetric product concentration were 1.38 and 2.55-fold higher compared to batch results, respectively. A preparative scale affinity chromatography resulted in a high recovery yield of 50.6 mg/L with ∼90 % purity. The conformational property of purified recombinant hIL-7 from CD spectroscopy showed a typical helical structure with 31.5 % α-helix and 26.43 % ß-sheet. The biological activity of purified protein was tested using IL-7-dependent murine immature B lymphocyte (2E8) cell line by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide salt (MTT) assay, where it showed a similar biological activity as standard control.

Comparison of the purification of biologically active IL-7 cytokine expressed in Escherichia coli and Pichia pastoris.

The large scale screening of cytokine mutants is a component of binding and activity mapping and requires an efficient method of cytokine protein expression. Here, we compared recombinant IL-7 expression with and purification from Escherichia coli and Pichia pastoris. The IL-7 cytokine contains three disulfide bonds that are essential for its biological activity, and which are formed upon secretion through P. pastoris, but not in the reducing cytoplasm of E. coli. In contrast to a previous report we found that P. pastoris secretes active but N-linked hyperglycosylated IL-7. Enzymatic deglycosylation was incompatible with activity measurements in a cell based assay. E. coli expressed IL-7 was refolded from solubilized inclusion bodies. A chromatographic purification step between inclusion body solubilization and refolding increased the yield of biologically active monomeric IL-7, and decreased the amount of inactive soluble aggregates. Cation exchange chromatography of untagged IL-7, and IMAC of His-tagged IL-7 improved refolding yields to a similar extend, indicating that the removal of contaminating components in the solubilized inclusion bodies improves refolding efficiency. We conclude that a chromatographic purification step of IL-7 solubilized from E. coli inclusion bodies increases refolding yield, and may be a suitable general rescue strategy for obtaining folded and biologically active proteins from inclusion bodies.

Lectin glycoprofiling of recombinant therapeutic interleukin-7.

Lectins array is a powerfull and complementary method of glycans analysis allowing fast identification of specific motifs on molecules or cells. This technology is of increased interest for the development of therapeutic recombinant glycoproteins and particularly relevant for a first study of lot-to-lot comparison, or detection of unwanted glycans. In this chapter, we describe a lectin array-type method specifically designed for the study of recombinant therapeutic interleukin-7 (rhIL-7). This specific method allows the analysis of the glycans motifs, the distribution of the glycoforms population, and the detection of potential immunogen glycans in rhIL-7 purified CHO-produced batches.

Structural insights into the common γ-chain family of cytokines and receptors from the interleukin-7 pathway.

Over the past 13 years, numerous crystal structures of complexes of the common γ-chain (γ(c)) cytokine receptors and their cytokines have been solved. Even with the remarkable progress in the structural biology of γ(c) receptors and their cytokines or interleukins, there are valuable lessons to be learned from the structural and biophysical studies of interleukin-7 (IL-7) and its α-receptor (IL-7Rα) and comparisons with other γ(c) family members. The structure of the IL-7/IL-7Rα complex teaches that interfaces between the γ(c) interleukins and their receptors can vary in size, polarity, and specificity, and that significant conformational changes might be necessary for complexes of interleukins and their receptors to bind the shared, activating γ(c) receptor. Binding, kinetic, and thermodynamic studies of IL-7 and IL-7Rα show that glycosylation and electrostatics can be important to interactions between interleukins and their receptor, even where the glycans and charged residues are distant from the interface. The structure of the IL-7Rα homodimer is a reminder that often-ignored non-activating complexes likely perform roles just as important to signaling as activating complexes. And last but not least, the structural and biophysical studies help explain and potentially treat the diseases caused by aberrant IL-7 signaling.

Interleukin-2, Interleukin-7, T cell-mediated autoimmunity, and N-glycosylation.

T cell activation and self-tolerance are tightly regulated to provide effective host defense against foreign pathogens while deflecting inappropriate autoimmune responses. Golgi Asn (N)-linked protein glycosylation coregulates homeostatic set points for T cell growth, differentiation, and self-tolerance to influence risk of autoimmune disorders such as multiple sclerosis (MS). Human autoimmunity is a complex trait that develops from intricate and poorly understood interactions between an individual's genetics and their environmental exposures. Recent evidence from our group suggests that in MS, additive and/or epistatic interactions between multiple genetic and environmental risk factors combine to dysregulate a common biochemical pathway, namely Golgi N-glycosylation. Here, we review the multiple regulatory mechanisms controlling N-glycan branching in T cells and autoimmunity, focusing on recent data implicating a critical role for interleukin-2 (IL-2) and IL-7 signaling.

Structural reorganization of the interleukin-7 signaling complex.

We report here an unliganded receptor structure in the common gamma-chain (γ(c)) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7Rα) extracellular domain (ECD) at 2.15 Å resolution reveals a homodimer forming an "X" geometry looking down onto the cell surface with the C termini of the two chains separated by 110 Å and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7Rα ECDs but a stronger association between the γ(c)/IL-7Rα ECDs, similar to previous studies of the full-length receptors on CD4(+) T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7Rα homodimer and IL-7Rα-γ(c) heterodimer to the active IL-7-IL-7Rα-γ(c) ternary complex whereby the two receptors undergo at least a 90° rotation away from the cell surface, moving the C termini of IL-7Rα and γ(c) from a distance of 110 Å to less than 30 Å at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7- and γ(c)-independent gain-of-function mutations in IL-7Rα from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other γ(c) receptors that form inactive homodimers and heterodimers independent of their cytokines.

Biophysical characterization of glycosaminoglycan-IL-7 interactions using SPR.

Glycosaminoglycans (GAGs) interact with a number of cytokines and growth factors thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and the regulation of the tissue distribution of cytokines/growth factors. In the present study, we employed surface plasmon resonance (SPR) spectroscopy to dissect the binding interactions between GAGs and murine and human forms of interleukin-7 (IL-7). SPR results revealed that heparin binds with higher affinity to human IL-7 than murine IL-7 through a different kinetic mechanism. The optimal oligosaccharide length of heparin for the interactions to human and murine IL-7 involves a sequence larger than a tetrasaccharide. These results further demonstrate that while IL-7 is principally a heparin/heparan sulfate binding protein, it also interacts with dermatan sulfate, chondroitin sulfates C, D, and E, indicating that this cytokine preferentially interacts with GAGs having a higher degree of sulfation.

Interleukin 7 from maternal milk crosses the intestinal barrier and modulates T-cell development in offspring.

BACKGROUND: Breastfeeding protects against illnesses and death in hazardous environments, an effect partly mediated by improved immune function. One hypothesis suggests that factors within milk supplement the inadequate immune response of the offspring, but this has not been able to account for a series of observations showing that factors within maternally derived milk may supplement the development of the immune system through a direct effect on the primary lymphoid organs. In a previous human study we reported evidence suggesting a link between IL-7 in breast milk and the thymic output of infants. Here we report evidence in mice of direct action of maternally-derived IL-7 on T cell development in the offspring. METHODS AND FINDINGS: We have used recombinant IL-7 labelled with a fluorescent dye to trace the movement in live mice of IL-7 from the stomach across the gut and into the lymphoid tissues. To validate the functional ability of maternally derived IL-7 we cross fostered IL-7 knock-out mice onto normal wild type mothers. Subsets of thymocytes and populations of peripheral T cells were significantly higher than those found in knock-out mice receiving milk from IL-7 knock-out mothers. CONCLUSIONS/SIGNIFICANCE: Our study provides direct evidence that interleukin 7, a factor which is critical in the development of T lymphocytes, when maternally derived can transfer across the intestine of the offspring, increase T cell production in the thymus and support the survival of T cells in the peripheral secondary lymphoid tissue.

In vivo antitumor activity of a recombinant IL-7/HGFbeta hybrid cytokine in mice.

The immune cytokine interleukin (IL)-7 and the ß-chain of hepatocyte growth factor (HGF) aggregate to form a naturally occurring heterodimer that stimulates the growth of common lymphoid progenitors and immature B and T lymphoid cells. We have cloned and expressed the heterodimer as a single-chain hybrid cytokine [recombinant (r) IL-7/HGFß], which stimulates short-term hematopoietic stem cells as well as lymphoid precursors. Inasmuch as IL-7 and HGF are known to have antitumor and protumor activities, respectively, we determined here whether either of these activities is exhibited by rIL-7/HGFß. We show that the in vivo administration of rIL-7/HGFß markedly inhibits the growth of newly initiated and established tumors and the formation of pulmonary metastases in murine models of colon cancer and melanoma. The antitumor effect of rIL-7/HGFß correlated with a marked increase in the number of tumor-infiltrating CD4(+) and CD8(+) T cells and activated dendritic cells. A major role for these immune cells in tumor suppression was indicated by the inability of rIL-7/HGFß to inhibit the growth of tumor cells in vitro and in congenitally athymic mice. Analysis of interferon-γ-secreting T cells showed that the immune response was tumor specific. Our findings justify further evaluation of rIL-7/HGFß as a novel experimental cancer therapy.

A biosensor study indicating that entropy, electrostatics, and receptor glycosylation drive the binding interaction between interleukin-7 and its receptor.

The interaction between interleukin-7 (IL-7) and its α-receptor, IL-7Rα, plays fundamental roles in the development, survival, and homeostasis of B- and T-cells. N-Linked glycosylation of human IL-7Rα enhances its binding affinity for human IL-7 300-fold versus that of the nonglycosylated receptor through an allosteric mechanism. The N-glycans of IL-7Rα do not participate directly in the binding interface with IL-7. This biophysical study involves dissection of the properties of binding of IL-7 to both nonglycosylated and glycosylated forms of the IL-7Rα extracellular domain (ECD) as functions of salt, pH, and temperature using surface plasmon resonance (SPR) spectroscopy. Interactions of IL-7 with both IL-7Rα variants display weaker binding affinities with increasing salt concentrations primarily reflected by changes in the first on rates of a two-step reaction pathway. The electrostatic parameter of the IL-7-IL-7Rα interaction is not driven by complementary charge interactions through residues at the binding interface or N-glycan composition of IL-7Rα, but presumably by favorable global charges of the two proteins. van't Hoff analysis indicates both IL-7-IL-7Rα interactions are driven by large favorable entropy changes and smaller unfavorable (nonglycosylated complex) and favorable (glycosylated complex) enthalpy changes. Eyring analysis of the IL-7-IL-7Rα interactions reveals different reaction pathways and barriers for the transition-state thermodynamics with the enthalpy and entropy changes of IL-7 binding to nonglycosylated and glycosylated IL-7Rα. There were no discernible heat capacity changes for the equilibrium or transition-state binding thermodynamics of the IL-7-IL-7Rα interactions. The results suggest that the unbound nonglycosylated IL-7Rα samples an extensive conformational landscape relative to the unbound glycosylated IL-7Rα, potentially explaining the switch from a "conformationally controlled" reaction (k(1) ∼ 10(2) M(-1) s(-1)) for the nonglycosylated interaction to a "diffusion-controlled" reaction (k(1) ∼ 10(6) M(-1) s(-1)) for the glycosylated interaction. Thus, a large favorable entropy change, a global favorable electrostatic component, and glycosylation of the receptor, albeit not at the interface, contribute significantly to the interaction between IL-7 and the IL-7Rα ECD.

CZE for glycoform profiling and quality assessment of recombinant human interleukin-7.

The aim of the present work was to develop a simple high-resolution CZE method for glycoform profiling and quality assessment of recombinant human interleukin-7 (rhIL-7) produced in Chinese hamster ovary cells. Classical and isoelectric buffers and several monoamines used as alkaline component of BGE at very low concentration have been investigated in order to optimize the resolution. The best separation was achieved using a fused-silica capillary and a buffer composed of 25 mM citrate/triethanolamine at pH 2.6. Under these acidic conditions, triethanolamine was able to reverse EOF favorably and to limit rhIL-7 adsorption. This method allowed the separation of four groups of peaks ranging from low to high-sialylated glycoforms. An extensive study on inter-run rinsing procedures has been conducted. Rinsing with 50 mM SDS was retained to achieve the optimal repeatability. Excellent intermediate precision was obtained for migration time (RSD < 0.6%), while RSD for intraday studies were only less than 2.9%. Satisfactory inter and intraday repeatabilities were also observed for relative peak area. We finally demonstrated that reliable information could be obtained to address comparability studies and demonstrate batch-to-batch consistency of biomanufactured rhIL-7.

Structural and biophysical studies of the human IL-7/IL-7Ralpha complex.

IL-7 and IL-7Ralpha bind the gamma(c) receptor, forming a complex crucial to several signaling cascades leading to the development and homeostasis of T and B cells. We report that the IL-7Ralpha ectodomain uses glycosylation to modulate its binding constants to IL-7, unlike the other receptors in the gamma(c) family. IL-7 binds glycosylated IL-7Ralpha 300-fold more tightly than unglycosylated IL-7Ralpha, and the enhanced affinity is attributed primarily to an accelerated on rate. Structural comparison of IL-7 in complex to both forms of IL-7Ralpha reveals that glycosylation does not participate directly in the binding interface. The SCID mutations of IL-7Ralpha locate outside the binding interface with IL-7, suggesting that the expressed mutations cause protein folding defects in IL-7Ralpha. The IL-7/IL-7Ralpha structures provide a window into the molecular recognition events of the IL-7 signaling cascade and provide sites to target for designing new therapeutics to treat IL-7-related diseases.

Crystallization and preliminary X-ray diffraction of human interleukin-7 bound to unglycosylated and glycosylated forms of its alpha-receptor.

The interleukin-7 (IL-7) signaling pathway plays an essential role in the development, proliferation and homeostasis of T and B cells in cell-mediated immunity. Understimulation and overstimulation of the IL-7 signaling pathway leads to severe combined immunodeficiency, autoimmune reactions, heart disease and cancers. Stimulation of the IL-7 pathway begins with IL-7 binding to its alpha-receptor, IL-7R alpha. Protein crystals of unglycosylated and glycosylated complexes of human IL-7-IL-7R alpha extracellular domain (ECD) obtained using a surface entropy-reduction approach diffract to 2.7 and 3.0 A, respectively. Anomalous dispersion methods will be used to solve the unglycosylated IL-7-IL-7R alpha ECD complex structure and this unglycosylated structure will then serve as a model in molecular-replacement attempts to solve the structure of the glycosylated IL-7-alpha-receptor complex.